Recombinant bivalent monospecific immunoglobulin having at least two variable fragments of heavy chains of an immunoglobulin devoid of light chains

ABSTRACT

The present invention relates to fragments, especially variable fragments of immunoglobulins which are by nature devoid of light chains, these fragments being nevertheless capable of exhibiting a recognition and binding activity toward specific antigens. The present invention further relates to the use of such immunoglobulin fragments formed of at least one heavy chain variable fragment or derived therefrom, for therapeutic or veterinary purposes and especially for passive immunotherapy or serotherapy.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/154,971, filed May 28, 2002, which is a continuation of U.S. patentapplication Ser. No. 08/945,244, filed Jan. 16, 1998, which is anational stage application under 35 U.S.C. §371 of InternationalApplication PCT/EP1996/01725 designating the United States of America,filed Apr. 25, 1996, the entire disclosures of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to fragments, especially variablefragments of immunoglobulins which are by nature devoid of light chains,these fragments being nevertheless capable of exhibiting a recognitionand binding activity toward specific antigens. These fragments ofimmunoglobulins can for example be obtained by the expression in hostcells for example in prokaryotic cells or eukaryotic cells of nucleotidesequences obtained from animals naturally expressing so-called“two-chain immunoglobulins”, for instance from animals of the camelidfamily.

The present invention further relates to the use of such immunoglobulinfragments formed of at least one heavy chain variable fragment orderived therefrom, for therapeutic or veterinary purposes and especiallyfor passive immunotherapy or serotherapy.

BACKGROUND OF THE INVENTION

Functional immunoglobulins devoid of light polypeptide chains termed<<two-chain immunoglobulin>> or <<heavy-chain immunoglobulin>> have beenobtained from animals of the family of camelids and have been describedin an international patent application published under number WO94/04678, together with two publications, especially Hamers-Casterman etal., 1993 and Muyldermans et al., 1994).

The isolation and characterization of these immunoglobulins, togetherwith their cloning and sequencing have been described in the abovereferenced documents which are incorporated by reference in the presentapplication.

According to WO 94/04678 it has been established that differentmolecules can be isolated from animals which naturally produce them,which molecules have functional properties of the well known four-chainimmunoglobulins these functions being in some cases related tostructural elements which are distinct from those involved in thefunction of four-chain immunoglobulins due for instance to the absenceof light chains.

These immunoglobulins having only two chains, neither correspond tofragments obtained for instance by the degradation in particular theenzymatic degradation of a natural four-chain model immunoglobulin, norcorrespond to the expression in host cells, of DNA coding for theconstant or the variable regions of a natural four-chain modelimmunoglobulin or a part of these regions, nor correspond to antibodiesproduced in lymphopathies for example in mice, rats or human.

The immunoglobulins devoid of light chains are such that the variabledomains of their heavy chains have properties differing from those ofthe four-chain immunoglobulin variable heavy chain (V_(H)). For clarityreasons, this variable domain according to the invention will be calledV_(HH) in this text to distinguish it from the classical V_(H) offour-chain immunoglobulins. The variable domain of a heavy-chainimmunoglobulin according to the invention has no normal interactionsites with the V_(L) or with the C_(H)1 domain which do not exist in theheavy-chain immunoglobulins. It is hence a novel fragment in many of itsproperties such as solubility and conformation of main chains. Indeedthe V_(HH) of the invention can adopt a three-dimensional organizationwhich distinguishes from the three-dimensional organization of knownfour-chain immunoglobulins according to the description which is givenby Chothia C. and Lesk A. M. (1987-J. Mol. Biol. 197, 901-917).

According to the results presented in patent application WO 94/04678,the antigen binding sites of the isolated immunoglobulins, naturallydevoid of light chains are located on the variable region of their heavychains. In most cases, each heavy chain variable region of thesetwo-chain immunoglobulins can comprise an antigen binding site.

A further characteristic of these two-chain immunoglobulins is thattheir heavy polypeptide chains contain a variable region (V_(HH)) and aconstant region (C_(H)) according to the definition of Roitt et al butare devoid of the first domain of the constant region is called C_(H)1.

These immunoglobulins of the type described hereabove can comprise typeG immunoglobulins and especially immunoglobulins which are termedimmunoglobulins of class 2 (IgG2) or immunoglobulins of class 3 (IgG3),according to the classification established in patent application WO94/04678 or in the publication of Muyldermans et al. (ProteinEngineering, Vol. 7, No. 9, pp 1129-1135-1994).

The absence of the light chain and of the first constant domain lead toa modification of the nomenclature of the immunoglobulin fragmentsobtained by enzymatic digestion, according to Roitt et al.

The terms Fc and pFc on the one hand, Fc′ and pFc′ on the other handcorresponding respectively to the papain and pepsin digestion fragmentsare maintained.

The terms Fab, F(ab)₂, F(ab′)₂, Fabc, Fd and fv are no longer applicablein their original sense as these fragments have either a light chain,the variable part of the light chain or the CH₁ domain.

The fragments obtained by papain digestion or by V8 digestion, composedof the V_(HH) domain of the hinge region will be called FV_(HH)h orF(V_(HH)h)₂ depending upon whether or not they remain linked by thedisulphide bonds.

The immunoglobulins referring to the hereabove given definitions can beoriginating from animals especially from animals of the camelid family.These heavy-chain immunoglobulins which are present in camelids are notassociated with a pathological situation which would induce theproduction of abnormal antibodies with respect to the four-chainimmunoglobulins. On the basis of a comparative study of old worldcamelids (Camelus bactrianus and Camelus dromaderius) and new worldcamelids (for example Lama Paccos, Lama Glama, and Lama Vicugna) theinventors have shown that the immunoglobulins devoid of lightpolypeptide chains are found in all species. Nevertheless differencesmay be apparent in molecular weight of these immunoglobulins dependingon the animals. Especially the molecular weight of a heavy chaincontained in these immunoglobulins can be from approximately 43 kd toapproximately 47 kd, in particular 45 kd.

Advantageously the heavy-chain immunoglobulins. of the invention aresecreted in blood of camelids.

The variable fragments of heavy chains of immunoglobulins devoid oflight chains can be prepared starting from immunoglobulins obtainable bypurification from serum of camelids according to the process for thepurification as described in detail in the examples of WO 94/04678. Thevariable fragments can also be obtained from heavy-chain immunoglobulinsby digestion with papain or V8 enzymes.

These fragments can also be generated in host cells by geneticengineering or by chemical synthesis. Appropriate host cells are forinstance bacteria (e.g. E. coli) eukaryotic cells including yeasts oranimal cells including mammalian cells, or plant cells.

The observation by the inventors that Camelidae produce a substantialproportion of their functional immunoglobulins as a homodimer of heavychains lacking the C_(H)1 domain and devoid of light chains(Hamers-Casterman et al., 1993), led to the proposal of having recourseto an immunized camel to generate and select single variable antibodyfragments (V_(HH)) and furthermore give access to the correspondingnucleotide sequences.

Cloned camel single V_(HH) fragments were displayed on bacteriophagesfor selection and in bacteria for the large scale production of thesoluble proteins, and were shown to possess a superior solubilitybehaviour and affinity properties compared to the mouse or human V_(H)equivalents (Muyldermans et al., 1994). Following this strategy, onewould obtain small ligand binding molecules (MW around 16,000 D) whichare not hindered by the presence of an oligopeptide linker (Borrebaecket al. 1992) or not inactivated by the disassembly of the VH-VL complex(Glockshuber et al., 1990). The camel V_(HH) fragments have theadditional advantage that they are characteristic of the heavy chainantibodies which are matured in vivo in the absence of light chains.

SUMMARY OF THE INVENTION

The inventors have obtained evidence that variable fragments of highchains of immunoglobulins devoid of light chains can display aneffective therapeutic activity when they are generated against adetermined antigen.

To develop this technology of preparing and identifying useful camelV_(HH) fragments, it is critical (i) that camels can be immunized with avariety of antigens, (ii) that the camel V_(HH) genes can be cloned andexpressed on filamenteous phages and in E coli for easy selection withthe immobilized antigen by panning, (iii) that the expressed camelV_(HH)'s are properly folded, and (iv) that they have good solubilityproperties and possess high affinities and specificities towards theirantigen.

Camel V_(HH) genes derived from the heavy chain immunoglobulins lackingthe light chains were previously cloned and analysed (Muyldermans etal., 1994). A comparison of the amino acid sequences of these camelV_(HH) clones clearly showed that the key features for preserving thecharacteristic immunoglobulin fold are all present. The specific aminoacid replacements observed in the camel V_(HH) clones could correlatewith the absence of the VL (variable light chains) and the functionalityof the camel single V_(HH) domain (Muyldermans et al., 1994).

The invention thus relates to a variable fragment (V_(HH)) of a heavychain of an immunoglobulin devoid of light chains, which is encoded by anucleotide sequence obtainable by the following process:

treating blood lymphocytes or other appropriate cells of an animal ofthe Camelid family previously immunized with a determined antigen, inorder to give access to their mRNA,

synthesizing a first strand of cDNA starting from the obtained mRNA,

contacting the obtained cDNA with at least two different primeroligonucleotides in conditions allowing their hybridization to at leasttwo complementary nucleotide sequences contained in the cDNA, saidprimers comprising a BACK primer (back p1) having nucleotide sequence(SEQ ID NO: 1): 5′-GATGTGCAGCTGCAGGCGTCTGG(A/G)GGAGG-3′ and a FOR primer(for p1) replying to the following nucleotide sequence (SEQ ID NOS: 2and 3, respectively: 5′-CGCCATCAAGGTACCGTTGA-3′ or5′-CGCCATCAAGGTACCAGTTGA-3′

amplifying the DNA fragment located between the nucleotide sequencehybridized with said primers and,

recovering amplified DNA corresponding to bands of different size ordersincluding:

-   -   a band of around 750 basepairs which is the amplified product of        the variable heavy chain (V_(H)), C_(H)1, hinge and part of CH2        region of a four-chain immunoglobulin,    -   a band of around 620 basepairs which is the amplified product of        the variable heavy-chain (V_(HH)), long hinge, and part of the        CH2 of the camel two-chain immunoglobulin IgG2,    -   a band of around 550 basepairs which is the amplified product of        the variable heavy-chain (V_(HH)), short hinge, and part of the        CH2 of the camel two-chain immunoglobulin IgG3,    -   purifying the two shortest bands of around 620 and 550 basepairs        from agarose gel, for example by Gene Clean,    -   recovering the amplified DNA fragments containing nucleotide        sequences encoding the V_(HH) fragments,    -   digesting the amplified products with restriction enzymes having        target sites within the amplified fragments and/or in the        nucleotide primers, for example with PstI and BstEII,    -   recovering the digested amplified DNA fragments,    -   ligating the amplified DNA fragments to a plasmid vector, for        example in a pHEN4 vector, in conditions allowing the expression        of the amplified fragments when the obtained recombinant vector        is used to transform a host cell,    -   transforming a determined bacterial host cell for example an E.        coli cell with the obtained recombinant plasmid vector, and        growing the cells on selective medium, to form a library,    -   infecting the obtained library of recombinant host cells after        culture in an appropriate selective medium, with bacteriophages,        for instance M13K07 bacteriophages to obtain recombinant        phagemid virions,    -   incubating the recombinant host cells in conditions allowing        secretion of recombinant phagemid virions particles containing        the recombinant plasmid, for instance the pHEN4 plasmid packaged        within the M13 virion.    -   isolating and concentrating the recombinant phagemid virions,    -   submitting the phagemid virions to several rounds of panning        with the antigen of interest previously immobilized, in        conditions allowing the adsorption of the phagemid virions on        the immobilized antigen,    -   eluting the adsorbed phagemid virions, and growing them on        appropriate cells,    -   amplifying the phagemid virions by infecting the cells with        helper bacteriophage,    -   recovering the virions and testing them for their binding        activity against the antigen of interest, for example by ELISA,    -   recovering the phagemid virions having the appropriate binding        activity,    -   isolating the nucleotide sequence contained in the plasmid        vector and capable of being expressed on the phagemid virions as        a V_(HH) amino acid sequence having the appropriate binding        activity.

In a preferred embodiment of the invention, the variable V_(HH)fragments are obtainable by adding to the hereabove describedamplification step of the cDNA with BACK and FOR primers (p1), a furtheramplification step with a BACK primer corresponding to theoligonucleotide sequence which has been described hereabove (back p1)and the FOR primer (for p2) having the following nucleotide sequence(SEQ ID NO: 4): 5′-CG ACT AGT GCG GCC GCG TGA GGA GAC GGT GAC CTG-3′.Not and BstEII sites which can be used for cloning in the pHEN4 vectorhave been underlined. This FOR primer allows hybridization to the codonposition of framework 4 (FR4) region of the V_(HH) nucleotide sequences(amino acid position 113-103).

According to another variant of the process described, this additionalamplification step can replace the amplification step which has beendescribed with BACK primer and a FOR primer having respectively thefollowing nucleotide sequences (SEQ ID NOS 1-3, respectively):5′-GATGTGCAGCTGCAGGCGTCTGG(A/G)GGAGG-3′

5′-CGCCATCAAGGTACCGTTGA-3′

or

5′-CGCCATCMGGTACCAGTTGA-3′

The restriction sites have been underlined.

In another embodiment of the invention the amplification step of thesynthetized cDNA is performed with oligonucleotide primers includinghereabove described BACK primer and FOR primer having the followingsequences (SEQ ID NOS 5 & 6, respectively): FOR primer 3: 5′-TGT CTT GGGTTC TGA GGA GAC GGT-3′

According to this latter embodiment, the V_(HH) fragments of theinvention are immediately and specifically amplified by a singleamplification (for instance PCR reaction) step when the mixture of FORprimers is used.

These latter primers hybridize with the hinge/framework 4 and shorthinge/framework 4 respectively. Each of these FOR primers allows theamplification of one IgG class according to the classification given inpatent application WO 94/04678.

The variable V_(HH) fragments corresponding to this definition can alsobe obtained from other sources of animal cells, providing that theseanimals are capable of naturally producing immunoglobulins devoid oflight chains according to those described in the previous patentapplication WO 94/04678.

These variable fragments (V_(HH)) can also be obtained by chemicalsynthesis or by genetic engineering starting from DNA sequences whichcan be obtained by the above described process.

The variable fragment of a heavy chain of an immunoglobulin devoid oflight chains according to the preceding definitions is specificallydirected against an antigen against which the animal has been previouslyimmunized, either by natural contact with this antigen or byadministration of this antigen in order to generate an immune responsedirected against it.

The process which is proposed hereabove to prepare a nucleotide sequencecoding for the variable fragments of the invention contains steps ofphage display library construction which allow the selection ofnucleotide sequences coding for variable fragments of heavy chainshaving the desired specificity.

According to one preferred embodiment of the invention, the variablefragments of a heavy chain of a immunoglobulin is obtainable from ananimal having been previously immunized with a toxin, especially a toxinof a bacteria or a part of this toxin sufficient to enable theproduction of immunoglobulins directed against this toxin and especiallyimmunoglobulins devoid of light chains.

According to another embodiment of the invention, the variable fragmentsof a heavy chain of a immunoglobulin is obtainable from an animal havingbeen previously immunized with substances contained in venom of animals.

The antigen used for immunization of the animals is usually under a nontoxic form.

The variable fragments according to the invention can be derived fromimmunoglobulins belonging to different classes especially belonging toIgG2 or IgG3 immunoglobulin classes, according to the classificationgiven in patent application WO/04678.

In a preferred embodiment of the invention, the variable fragment of aheavy-chain of an immunoglobulin devoid of light chains is directedagainst the tetanus toxin of Clostridium tetani or against a fragmentthereof.

The variable fragments of heavy chains of immunoglobulins devoid oflight chains can be also generated against toxins or part thereof frompathogenic organisms such as bacteria and especially can be chosen amongthe toxins or toxoids of the following bacteria: Clostridium, especiallyClostridium Botulinum or Clostridium Perfringens, Staphylococcus,Pseudomonas, Pasteurella, Yersinia, Bacillus Anthracis, Neisseria,Vibrio, especially Vibrio cholera, enterotoxic E. coli. Salmonella,Shigella, Listeria.

Other antigens appropriate for the preparation of the V_(HH) fragmentsof the invention can be obtained from the following organism: anemonies,coral, jellyfish, spiders, bees, wasps, scorpions, snakes, includingthose belonging to the families of Viperidae, Crotalidae, Lapidea.

According to another embodiment of the invention, the variable fragmentV_(HH) of a heavy chain of an immunoglobulin devoid of light chains ischaracterized in that it comprises the following amino acid sequences(SEQ ID NOS: 25 & 26, respectively):

(Glu/Asp) Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly(Gly/Gln) Ser Leu Arg Leu Ser Cys Ala (Ala/Thr) Ser Gly (CDR1) Trp(Phe/Tyr) Arg Gln Ala Pro Gly Lys Glu (Arg/Cys) Glu (Gly/Leu) Val(Ser/Ala) (CDR2) Arg (Phe/Leu) Thr Ile Ser (Arg/Leu/Gln) Asp Asn Ala LysAsn Thr (Val/Leu) Tyr Leu (Gln/Leu) Met Asn Ser Leu (Lys/Glu) Pro GluAsp Thr Ala (Val/Met/Ile) Tyr Tyr Cys Ala Ala (CDR3) Trp Gly Gln Gly ThrGln Val Thr Val Ser Ser or

(Glu/Asp) Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly(Gly/Gln) Ser Leu Arg Leu Ser Cys Ala (Ala/Tlu) Ser Gly(Ala,Thr,Ser,Ser/Tyr,Thr,Ile,Gly) (CDR1) Trp (Phe/Tyr) Arg Gln Ala ProGly Lys Glu (Agr/Cys) Glu (Gly/Leu) Val (Ser/Ala) (CDR2) Arg (Phe/Leu)Thr Ile Ser (Arg/Leu/Gln) Asp Asn Ala Lys Asn Thr (Val/Leu) Tyr Leu(Gln/Leu) Met Asn Ser Leu (Lys/Glu) Pro Glu Asp Thr Ala (Val/Met/Ile)Tyr Tyr Cys Ala Ala (CDR3) Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser,

wherein CDR1, CDR2 and CDR3 represent variable amino acid sequencesproviding for the recognition of a determined epitope of the antigenused for the immunization of Camelids, CDR1, CDR2 and CDR3 sequencescomprising from 5 to 25 amino acid residues preferably CDR1 containsfrom 7 to 12 amino acid residues, CDR2 contains from 16 to 21 amino acidresidues and CDR3 contains from 7 to 25 amino acid residues.

The camel V_(HH) specific amino acid residues Ser 11, Phe 37, Glu 44,Arg 45, Glu 46, Gly 47 are underlined.

One preferred variable fragment according to the invention is encoded bya nucleotide sequence present in recombinant plasmid pHEN4-αTT2(WK6)deposited at the BCCM/LMBP (Belgium) under accession number LMBP3247.

The pHEN4-αTT2 (described in FIG. 2) is a plasmid carrying a PeIB leadersignal, a camel V_(HH) gene of which the protein binds tetanus toxoid, adecapeptide tag (frim immunoZAP H of Stratacyte) and gene IIIp of M13 inthe pUC 119 polylinker between the HindIII and EcoR1 sites. This plasmidwas transfused in E. coli WK6 cells.

A specific variable fragment according to the invention is for instancecharacterized in that it comprises the following αTT1 amino acidsequence (SEQ ID NO: 7):

Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Gly Gln Thr Phe Asp Ser Tyr Ala Met AlaTrp Phe Arg Gln Ala Pro Gly Lys Glu Cys Glu Leu Val Ser Ser Ile Ile GlyAsp Asp Asn Arg Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asp Arg Leu Asn Pro Glu AspThr Ala Val Tyr Tyr Cys Ala Gln Leu Gly Ser Ala Arg Ser Ala Met Tyr CysAla Gly Gln Gly Thr Gln Val Thr Val Ser Ser

According to another preferred embodiment of the present invention, thevariable fragment comprises the following αTT2 amino acid sequence (SEQID NO: 8):

Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser LeuArg Leu Ser Cys Thr Ala Ala Asn Tyr Ala Phe Asp Ser Lys Thr Val Gly TrpPhe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Gly Ile Ser Ser GlyGly Ser Thr Thr Ala Tyr Ser Asp Ser Val Lys Gly Arg Tyr Thr Val Ser LeuGlu Asn Ala Lys Asn Thr Val Tyr Leu Leu Ile Asp Asn Leu Gln Pro Glu AspThr Ala Ile Tyr Tyr Cys Ala Gly Val Ser Gly Trp Arg Gly Arg Gln Trp LeuLeu Leu Ala Glu Thr Tyr Arg Phe Trp Gly Gln Gly Thr Gln Val Thr Val SerSer

In a preferred embodiment of the invention, the variable V_(HH) fragmentof the invention is altered in order to diminish its immunogenicproperties. Such a modification can lead to an alternated immunologicalreaction against the V_(HH) fragments of the invention when they areadministered to a host either human or animal, for passiveimmunoprotection for example.

The invention further relates to a pharmaceutical composition comprisingan immunoglobulin heavy chain variable fragment according to those whichhave been defined hereabove, in admixture with a physiologicallyacceptable vehicle.

Such pharmaceutical composition can be used for the treatment by passiveimmunisation, of infections or acute intoxications by toxins such asthose of Clostridium, especially Clostridium Botulinum or ClostridiumPerfringens, Staphylococcus, Pseudomonas, Pasteurella, Yersinia,Bacillus Anthracis, Neisseria, Vibrio, especially Vibrio cholera,enterotoxic E. coli, Salmonella, Shigella, Listeria or anemonies, coral,jellyfish, spiders, beas, wasps, scorpions, snakes, including thosebelonging to the families of Viperidae, Crotalidae, Lapidea.

The present invention further relates to nucleotide sequences coding fora variable fragment (V_(HH)) of a heavy chain of an immunoglobulindevoid of light chains, obtainable by the process which has beendescribed hereabove.

Specific nucleotide sequences are those corresponding to αTT1 and αTT2as described on FIGS. 4A and 4B.

According to an embodiment of the invention, a preferred nucleotidesequence is the sequence contained on plasmid pHEN4-αTT2 deposited atthe BCCM/LMBP collection in Belgium on Jan. 31, 1995 under no. LMBP3247.

The invention further provides means for the preparation of bivalent oreven multivalent monospecific DNA constructs of variable fragments of animmunoglobulin devoid of light chains and their expression products. Itthus gives access to the preparation of monovalent bispecific ormultispecific variable constructs obtained from sequences encodingV_(HH) fragments combined with a linker sequence. Bivalent monospecificconstructs contain 2 nucleotide sequences coding for V_(HH) fragmentsdirected against the same antigen or epitope. Monovalent bispecificconstructs contain on one molecule one nucleotide sequence coding for aV_(HH) fragment directed against one antigen or epitope and anothernucleotide sequence coding for a fragment directed against anotherantigen or epitope.

The corresponding expression products (protein constructs) can beobtained by genetic engineering especially by expression in host cells,like bacteria (e.g. E. coli) or eukaryotic cells, of the above DNAconstructs.

Accordingly a variable fragment of the V_(HH) type having a determinedantigen specificity, can be linked to at least one further variablefragment V_(HH) having a determined similar or different specificity interms of antigen and/or epitope specificity.

The obtained constructs (in terms of expression products) and especiallythe bivalent monospecific constructs advantageously offer means toimprove the affinity for the antigen(s) against which they are obtained.

The linker sequence between the V_(HH) fragments can be for example asequence corresponding to the coding sequence of the hinge domain ofimmunoglobulin devoid of light chains (e.g. the long hinge domain) asdescribed by (Hamers-Casterman C. et al, 1993) or a sequence derivedtherefrom.

As an example, in order to ligate these two variable coding sequences ofV_(HH) fragments to obtain monovalent bispecific construct, the sequencecoding for the hinge and CH₂ domains, especially coding for the longhinge and CH₂ domains of an immunoglobulin devoid of light chains can beused. These domains have been described in WO 94/04678.

As another example, for instance for the preparation of bispecific ormultispecific DNA constructs, the sequence used as linker between theV_(HH) fragments is derived from the coding sequence of the hinge and isdevoid of the terminal part containing nucleotides coding for thecysteine residue, or more generally devoid of the codons enablingdimerisation of the V_(HH) fragment.

Preferred linkers include: the sequence starting at nucleotide 400 andending at nucleotide 479 or between nucleotides 479 and 486 of thenucleotide sequence disclosed on FIG. 15 or the sequence starting atnucleotide 400 and ending at nucleotide 495 or between nucleotides 487and 495 of the nucleotide sequence of FIG. 15.

The linkers can be for instance obtained by digestion of a plasmidcontaining the coding sequence for the V_(HH), hinge and CH₂ domains ofan immunoglobulin devoid of light chains, with BstEII and XmnI (or KpnI)endonucleases and further amplification of the sequence with primersannealing to each end of the hinge coding sequence as described aboveand illustrated in the examples.

As an example, constructs (monovalent or multivalent, monospecific ormultispecific) can be obtained having a specificity with respect to twoor more different toxins or generally antigens of different pathogenorganisms including bacteria and viruses.

The invention also relates to a process for the preparation ofmonovalent bispecific constructs of variable fragments of a heavy chainof an immunoglobulins which comprises the following steps:

a) ligating a nucleotide sequence coding for a variable V_(HH) fragmenthaving a determined antigen- or epitope-specificity to a linkernucleotide sequence to form a V_(HH) linker fragment;

b) ligating the formed nucleotide sequence coding for the V_(HH)-linkerfragment to a nucleotide sequence coding for another V_(HH) fragmenthaving a different antigen- and/or epitope-specificity,

wherein the linker sequence contains the nucleotide sequence coding forpart of a hinge domain wherein the codons responsible for thedimerisation of the V_(HH) fragments especially by formation of adisulfide bridge between the last cysteine residues within the hingedomain are deleted.

According to a preferred embodiment, additional steps of ligation areperformed with sequences coding for variable fragments (V_(HH)fragments) having the same specificity or a different specificity withrespect to the above fragments.

In such a case the V_(HH) fragment coding sequences recovered from stepb) must be digested so as to produce a nucleotide sequence having thefollowing structure hinge linker-V_(HH). In accordance V_(HH)-hingelinker)_(n) coding sequences are obtained wherein n is a number higherthan 2.

Preferably, the sequence encoding the hinge domain preferably the longhinge domain of the immunoglobulins devoid of light chains is thenucleotide sequence comprising or corresponding to nucleotides 400 to479 or up to nucleotides 486 of the sequence of FIG. 15.

In a particular embodiment of the process for the preparation ofbivalent or multivalent monospecific or multispecific constructs, theV_(HH) fragment coding sequence linked to a nucleotide sequence encodingthe hinge domain has to be amplified. Oligonucleotide primers have beendefined which permit the amplification of the sequence of interest.These oligonucleotides anneal respectively with their 3′ end to thebeginning of the V_(HH) gene or coding sequence and to the terminal partof the hinge coding sequence. Appropriate primers are for instance (SEQID NOS: 9 & 10, respectively):

A4 (Sf I site underlined):5′-CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCGA(G,T)GT(G, C)CAGCT-3′

AM007: 5′-GGCCATTTGCGGCCGCATTCCATGGGTTCAGGTTTTGG-3′

These chosen primers contain target sequences for specificendonucleases, thus allowing the cloning of the digestion products ofthe amplified fragments in a suitable vector.

The obtained DNA constructs are then used to transform host cells, forinstance E. coli and the expressed proteins are then isolated andpurified. The expression products of these DNA constructs are within thescope of the invention.

The heavy-chain antibodies, such as those derived from camel, and theirfragments present clear advantages over other antibodies or fragmentsthereof derived from other animals. These are linked to the distinctivefeatures of the heavy chain antibodies and in particular the novelfragments which can be produced by proteolytic cleavage within the hingeof these heavy-chain antibodies to generate the V_(HH) and the(V_(HH)h)2 fragments. The V_(HH) domain of a heavy chain has distinctgenetic entities which confer properties of solubility not found in VHfragments derived from conventional antibodies. This property, inaddition to its small size and to the fact that the amino acid sequenceof the framework region is very homologous to that of human, ensures aminimum of immunogenicity. These properties would allow repetitivetreatment with heavy chain V_(HH) fragments for passive immunisation orantibody therapy. As mentioned above, V_(HH) and the (V_(HH)h)2fragments can easily be produced by proteolytic cleavage of camelimmunoglobulins or via recombinant DNA technology.

The most important field of passive immunisation is intoxication due tobacterial toxins and in particular acute intoxication or intoxicationdue to drug resistant bacteria. Passive immunisation or treatment byantibodies is justified in those cases where vaccination is unpracticalor its effects short-lived. They are particularly justified for acuteintoxication which if left untreated would have lethal or cripplingeffects.

The following list of indications is non-exhaustive:

Tetanos due to infection by Clostridium tetani is an importantpost-trauma infection and current immunisations are not long lasting. Itis also important in the veterinary field.

Botulism due to ingestion of toxins produced by Clostridium Botulinumand related species.

Gangrene due to infection by Clostridium.

Necrotic Enteritis and Enterotoxemia in humans and livestock due toClostridium Perfringens ingestion.

Food poisoning due to Staphylococcal endotoxins in those cases whereantibiotics are not recommended.

Pseudomonas infection refractory to antibiotic treatment and inparticular ocular infections where rapid intervention is warranted.

Diphteria toxin infection

Pasteurella and Yersinia infection causing lethal outcomes in human andlivestock.

Anthrax toxin produced by Bacillus Anthraxis and responsible for one ofthe five major livestock diseases.

Infections due to other bacterial agents such as Neisseria or viralagents.

Furthermore, the relative resistance of the V_(HH) fragment toproteolytic cleavage by digestive enzymes (e.g. pepsin, trypsin) offerthe possibility of treatment against important gut pathogens, such asVibrio cholera and other vibrios, enterotoxic E. coli, Salmonellaspecies and Shigella or pathogens ingested with food such as Listeria.

Another major target for immunotherapy is in the treatment ofintoxication due to bites or contact with toxic invertebrates andvertebrates. Among the invertebrates are sea anemonies, coral andjellyfish, spiders, bees and wasps, scorpions. In the vertebrates, thevenemous snakes are of particular importance and in particular thosebelonging to the families of Viperidae, Crotalidae and Lapidea.

Passive immunisation with partially purified immunoglobulins fromimmunized animals are already being used. In developing countries,antitetanos and antidiphteria antisera are still produced on a verylarge scale, usually in horses. Anti-venom antibodies are produced,although on a much smaller scale, against venoms, especially snakevenoms.

Another field of application is in combination with the therapeutic useof toxins in medical or surgical practice where neurotoxins such asbotulinum toxin are increasingly used.

The invention also relates to the oligonucleotide primers describedhereabove, either alone or in kits.

Other characteristics of the invention will appear from the figures andthe examples which are described hereafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: 1% agarose gel electrophoresis of the PstI/BstEII digested PCRamplification product of the camel V_(HH) gene (lanes 1 and 2) next tothe 123 bp ladder of BRL used as a size marker (lane 4). The PCR productcomigrates with the 3^(rd) band of the marker, 369 bp in length.

FIG. 2: Map of the pHEN4 with the nucleotide sequence of the V_(HH)cloning site shown in the lower part of the figure (SEQ ID NOS: 27-29,respectively). The PstI and BstEII sites can be used to clone the camelV_(HH) PCR product shown in FIG. 1.

FIG. 3: 100 individual clones were randomly selected from the originalcamel V_(HH) library (O), or after the first (1), second (2), third (3)or fourth (4) round of panning. After M13 infection the virions weretested for binding activity against immobilized tetanus toxoid. Thenumber of positive clones are shown as a function of number of pannings.

FIGS. 4A and 4B: Nucleotide sequence and the corresponding amino acidsequence of the two identified camel V_(HH) anti tetanus toxoid clonespHEN4-αTT1 (FIG. 4A) and pHEN4-αTT2 (FIG. 4B) (SEQ ID NOS: 13 & 14 and15 & 16, respectively). The framework Serll, Phe37 and kg or Cys 45characteristic for the camel V_(HH) heavy chain antibodies (Muyldermanset al., 1994) are double underlined.

FIG. 5: SDS-polyacrylamide gel electrophoresis of the proteins extractedfrom the periplasm of WK6 cultures induced with IPTG. Lane 1 & 8,protein size marker (Pharmacia) MW are (from top of to bottom) 94,000;67,000; 43,000; 30,000; 20,100 and 14,400 D. Lanes 2 and 7 Expressedperiplasmic proteins extracted from WK6 cells containing pHEN4-αTT2′ andpHENA-αTT1′ cloning vector. Lane 3 & 4, Purified V_(HH) domain ofpHEN4-αTT2 at 10 and 1 microgram. Lanes 5 & 6, Purified V_(HH) domain ofpHEN4-αTT1 at 10 and 1 migrogram. The position of the expressed solublecamel VH protein is indicated with an arrow. It is clearly absent in thesecond lane.

FIG. 6: The total periplasmic extract of 1 liter of culture of WK6 cellscarrying the pHEN4-αTT2 was concentrated to 5 ml and fractionated by gelfiltration on Superdex 75 (Pharmacia) using 150 mM NaCL, 10 mM sodiumphosphate pH 7.2 as eluent. The pure V_(HH) is eluted at the fractionsbetween the arrows.

FIG. 7: CD (Circular dichroism) spectrum (Absorbance versus wavelengthin nm) of the purified V_(HH) domain αTT2 at 3.9×10⁻⁶ Min water measuredin a cuvette with a path length of 0.2 cm. The negative band near 217and 180 nm and the positive band around 195 nm are characteristic for βstructures (Johnson, 1990).

FIG. 8: Specificity of antigen binding shown by competitive ELISA. Theexperiments were carried out in triplicate with the bacterialperiplasmic extracts of pHEN4-αTT1 and pHEN4-αTT2.

FIG. 9: Number of mice surviving after I.P injection of 100 ngr tetanustoxin (10×LD50) or co-injection of tetanus toxin with the purifiedV_(HH) αTT1, αTT2 or the non-specific cVH21 (Muyldermans et al., 1994)at 4 or 40 microgram.

FIG. 10: Variability plot of the camelid V_(HH) sequence (CDR3 andframework 4 regions are not included).

The alignment of the V_(HH) amino acid sequences of camel and lama (atotal of 45 sequences) was performed according to Kabat et al. Thevariability at each position was calculated as the number of differentamino acids occurring at a given position, divided by the frequency ofthe most common amino acid at that position. Positions are numberedaccording to Kabat et al. The positions above the horizontal barindicate the amino acids which are referred to as (CDR1) and (CDR2) inthe consensus sequence.

A variability number equal to 1 indicates a perfectly conserved aminoacid at that position. The higher the variability number the more likelyit will be that the amino acid at this position will deviate from theconsensus sequence.

FIG. 11: Nucleic acid sequence of LYS2 V_(HH) and translation productthereof (SEQ ID NOS: 17 & 18, respectively).

FIG. 12: Nucleic acid sequence of LYS3 V_(HH) and translation productthereof (SEQ ID NOS: 23 & 24, respectively).

FIG. 13A: Scheme to construct the bivalent monospecific anti-LYS3 camelV_(HH).

FIG. 13B: Scheme of constructed monovalent bispecific andi-LYS3-longhinge linker-anti-LYS2-Tag and schematic diagram of same.

FIG. 14A: Scheme of constructed bispecific anti-LYS3-long hingelinker-anti-LYS2-Tag and schematic diagram of same.

FIG. 14B: Scheme of constructed monovalent bispecific anti-LYS3-longhinge linker-anti-LYS-2 Tag and schematic diagram of same.

FIGS. 15A and 15B: Nucleotide and amino acid sequence (SEQ ID NOS: 21 &22, respectively) of the anti-LYS3-long hinge/Cys-Tag. This protein willspontaneously dimerise.

FIGS. 16A, 16B and 16C: Nucleotide and amino acid sequence (SEQ ID NOS:23 & 24, respectively) of the anti-LYS3-long hinge linker-anti-LYS2-Tagpolypeptide.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE I

Generation of Specific Camel V_(HH) Fragments Against Tetanus Toxoid

In this application, results are presented, which prove the feasibilityof generating specific camel V_(HH) fragments with demonstrated foldingand good binding affinity. This was done by generating a library ofcamel fragments derived from the dromedary IgG2 and IgG3 isotype,display of the V_(HH) library on phage as fusion proteins with the geneIII protein of bacteriophage M13 to allow selection of the antigenbinders, and finally of expressing and extracting the soluble andfunctional V_(HH) fragments from E. coli. As antigen, we choose thetetanus toxoid was chosen because comparisons are possible withpublished data. In addition, the tetanus toxoid is a highly immunogenicprotein that is routinely used as a vaccine in humans to elicitneutralizing antibodies. The two camel V_(HH) fragments that wereidentified were specific and of high affinity. The affinities of the twocamel V_(HH) fragments appear to be comparable with those from the humananti-tetanus toxoid F_(AB)'S recently obtained by Mullinas et al. (1990)and by Persson et al. (1991).

Camel Immunization

The serum of a camel (Camelus dromedarius) was shown to be non-reactingwith tetanus toxoid (RIT, Smith Kline Beecham, Rixensart, Belgium). Thiscamel was injected with 100 μg tetanus toxoid at days 9, 30, 52, 90 andwith 50 μg at days 220, 293 and 449. The blood was collected 3 daysafter each injection.

mRNA Purification of Camel Blood Lymphocytes

Peripheral blood lymphocytes were purified with Lymphoprep (Nycomed,Pharma) from the bleeding at day 452. Aliquots of 1×10⁶−5×10⁶ cells werepelleted and frozen at −85° C. and subsequently used as an enrichedsource of B-cell mRNA for anti-tetanus toxoid.

The mRNA was prepared from a total of 106 peripheral blood lymphocyteseither by the “Micro FastTrack” mRNA isolation kit (Invitrogen) or the“QuickPrep Micro mRNA Purification” kit of Pharmacia, following therecommendations of the manufacturer. With both protocols, up to a fewμgr of mRNA was obtained which was used in the subsequent cDNA synthesisstep.

cDNA Synthesis and PCR Amplification of Camel V_(HH) Gene

The first-strand cDNA was synthesized with the Invitrogen “cDNA-cycle”or the Pharmacia “Ready-To-Go” kit. The first-strand cDNA was usedimmediately afterwards for the specific amplification of the camelV_(HH) region by PCR. The primers used have following sequences: theBACK primer (SEQ ID NO: 1) (5′-GATGTGCAGCTGCAGGCGTCTGG(A/G)GGAGG-3′),the internal PstI site is underlined) is designed to hybridize to theframework 1 region (codons 1 to 10) of the camel V_(HH), while the FORprimer (5′-CGCCATCMGGTACCAGTTGA-3′) hybridizes in the CH2 region. ThePCR was carried out with the Taq polymerase from Boehringer Mannheim.

The PCR product was purified according to standard protocols (Sambrooket al., 1989) and digested with the PstI restriction enzyme of which thetarget site occurred in the BACK primer, and with BstEII which has anaturally occurring site in the framework 4 of the camel V_(HH) regions.The resulting fragments of approximately 360 bp (FIG. 1) were ligatedinto the pHEN4 vector cut with the same restriction enzymes. The pHEN4vector (FIG. 2) is the pHEN1 plasmid (Hoogenboom et al., 1991)—a pUC119based vector—where the myc-tag was replaced by the decapeptide tagpresent in the ImmunoZAP H vector (Stratacyte). Also the polylinker wasmodified to allow the cloning of the camel V_(HH) gene between a PstIand a BstEII site located after the PeIB leader signal and in front ofthe decapeptide tag and gene III of bacteriophage M13.

Construction of a Camel V_(HH) Library

The ligated DNA material was precipitated with 10 volumes andresuspended in 10 μl water and electrotransformed in E. coli XL1 BlueMRF′ cells (Stratagene). After electroporation according to therecommended protocol (Stratagene) we kept the cells for 1 hour at 37° C.in 1 ml SOC medium before plating on LB plates containing 100 μgampicilline/ml. After an over night incubation at 37° C. the transformedcells were grown out into colonies and some 500,000 recombinant cloneswere obtained. About 20 colonies, randomly selected, were toothpickedand grown in selective medium (LB/Ampicilline) to prepare plasmid DNAand to check their insert by sequencing. For each clone tested, we founda different V_(HH) region with the amino acid sequence and contentscharacteristic for a V_(HH) originating from a camel heavy chainimmunoglobulin (Muyldermans et al., 1994). This indicates that a vastcamel V_(HH) library was generated.

The remaining 500,000 clones were scraped from the plates with a minimalamount of LB containing 50% glycerol and stored at −85° C. until furtheruse.

Panning with Tetanus Toxoid

The library was screened for the presence of anti-tetanus toxoid camelV_(HH)'s by panning. To this end, approximately 10⁹ cells (=5 mlsuspension of the frozen recombinant clones) were grown tomidlogarithmic phase in 200 ml of LB medium supplemented with 1% glucoseand 100 μg ampicilline/ml before infection with M13K07 bacteriophages.After adsorption of the bacteriophages on the E. coli cells for 30 minat room temperature, the cells were harvested by centrifugation andwashed in LB medium supplemented with ampicilline and kanamycin (25μg/ml). The cells were incubated overnight at 37° C. to secrete therecombinant pHEN plasmid packaged within the M13virion containing acamel V_(HH) fused to some of its M13gene III proteins (Hoogenboom etal., 1991). The phagemid virions were prepared according to the protocoldescribed by Barbas et al. (1991). The phage pellets were resuspended inblocking solution (1% casein in phosphate buffered saline, PBS),filtered through a 0.2 μm filter into a sterile tube and used forpanning. For the panning the Falcon 3046′ plates were coated overnightwith 0.25 mg/ml or 2 mg/ml tetanus toxoid dissolved in PBS orhydrogencarbonate pH 9.6. The wells were subsequently washed andresidual protein binding sites were blocked with blocking solution atroom temperature for 2 hours. The adsorption of the phagemid virions onthe immobilized antigen and the washing and elution conditions wereaccording to Marks et al. (1991) or were taken from the protocoldescribed by the <<Recombinant Phage Antibody System>> of Pharmacia.Four consecutive rounds of panning were performed. After the fourthround of panning the eluted phagemid virions were added to exponentiallygrowing TGI cells (Hoogenboom et al. 1991) and plated on ampicillincontaining LB plates. After overnight growth several colonies were grownindividually in LB medium to midlogarithmic growing phase, and infectedwith M13K07 helper phage. The virions were prepared and tested for theirbinding activity against tetanus toxid immobilised on mitrotiter plates.The presence of the virion binding to the immobilized antigen wasrevealed by ELISA using a Horse RadishPeroxidase/anti-M13 conjugate(Pharmacia). The percentage of binders was increasing after each roundof pannning. In the original library we found 3 clones out of 96 whichshowed binding with the immobilized tetanus toxoid. This number wasincreased to 11, 48 and 80 after the first, second and third round ofpanning. All of the individual clones which were tested after the fourthround of panning were capable of recognizing the antigen, as measured byELISA (FIG. 3). Ten positive clones were grown and tested by PCR tocheck the presence of an insert with the proper size of the V_(HH) gene,and their DNA was finally sequenced. The sequencing data revealed thattwo different clones were present among this set of 10 clones. Theplasmid DNA of these clones was named pHEN4-αTT1 and pHEN4-αTT2. PlasmidDNA was deposited at the “Belgian Coordinated Collections ofMicroorganisms” BCCM/LMBP on Jan. 31, 1995 under accession numberLMBP3247, and it was shown that these two different clones contained acDNA coding for a camel V_(HH) (FIG. 4). Comparison of the amino acidsin these clones with the camel V_(HH) clones analysed before(Muyldermans et al., 1994) clearly indicated that the anti-tetanus camelV_(HH) originated from a heavy chain immunoglobulin lack of the CH1domain and light chains. Especially the identity of the key residues atposition 11 (Ser), 37 (Phe) and 45 (Arg or Cys) and 47 (Leu or Gly)proved this statement (Muyldermans et al., 1994).

Production of Soluble Camel V_(HH) with Anti-Tetanus Toxoid Activity

The plasmid DNA of the two clones which scored positive in the tetanustoxoid ELISA were transformed into WK6 cells. These cells are unable tosuppress the stopcodon present in the vector between the decapeptide tagand the gene III protein. The WK6 E. coli cells harboring the pHEN4-αTT1or pHEN4-αTT2 plasmid were grown at 37° C. in 1 liter of TB medium with100 mgr ampicillin/ml and 0.1% glucose. When the cells reached an OD₅₅₀of 1.0 we harvested the cells by centrifugation at 5000 rpm, 10 minutes.The cell pellet was washed once in TB medium with ampicillin, butomitting the glucose. The cells were finally resuspended in 1 liter ofTB medium with ampicillin (100 μg/ml). We induced the expression of thecamel V_(HH) domain by the addition of 1 mM IPTG and further growth ofthe cells at 28° C. for 16 hours. The expressed proteins were extractedfrom the periplasmic space following the protocol described by Skerraand Plucthun (1988). We pelleted the E. coli cells by centrifugation at4000 g for 10 min. (4° C.). The cells were resuspended in 10 ml TESbuffer (0.2 M Tris-HCl pH 8.0, 0.5 mM EDTA, 0.5 M sucrose). Thesuspension was kept on ice for 2 hours. The periplasmic proteins wereremoved by osmotic shock by addition of 20 ml TES diluted ¼ with water.The suspension was kept on ice for 1 hour and subsequently centrifugedat 12,000 g for 30 minutes at 4° C. The supernatant contained theexpressed camel V_(HH) domain. The extract corresponding to 400 μl cellculture was applied under reducing conditions on a SDS/polyacrylamideprotein gel. The extracted proteins were visualized in theSDS/polyacrylamide gels by Coomassie blue staining (FIG. 5). A proteinband with an apparent molecular weight of 16,000 D was clearly presentin the E. coli cultures containing the recombinant clones and inducedwith IPTG. Alternatively, the presence of the camel V_(HH) proteins inthe extract was revealed by Western blot using a specific rabbitanti-camel V_(HH) or rabbit anti-dromedary IgG serum or the anti-tagantibody.

We estimate from the band intensity observed in the Coomassie stainedgel that more than 10 mg of the camel V_(HH) protein (non-purified) canbe extracted from the periplasm of 1 liter induced E. coli cells.

For the purification of the anti-tetanus toxoid camel V_(HH) weconcentrated the periplasmic extract 10 times by ultrafiltration(Millipore membrane with a cut off of 5000 Da). After filtration theconcentrated extract from the pHEN4-αTT2 was separated according itsmolecular weight by gelfiltration on Superdex-75 (Pharmacia) (FIG. 6)equilibrated with PBS (10 mM phosphate buffer pH 7.2, 150 mM NaCl). Thepeak containing the anti-tetanus toxoid activity eluted at the expectedmolecular weight of 16,000 Da indicating that the protein behaved as amonomer and does not dimerize in solution. The fractions containing thepure V_(HH) (as determined by SDS-PAGE) were pooled and theconcentration was measured spectrophotometrically using a calculatedE₂₈₀ (0.1%) of 1.2 and 2.3 respectively for the αTT1 and αTT2. From theUV absorption at 280 nm of the pooled fraction we could calculate ayield of 6 mgr of purified protein per liter of bacterial culture. Thepurified protein could be further concentrated by ultrafiltration to 6mgr/ml in PBS or water without any sign of aggregation, as seen on theUV spectrum.

Concerning the expression yield in E. coli it should be realized that atthis stage we did not try to optimize the expression or the proteinextraction conditions. However, as the yield of the purified αTT2 camelV_(HH) reached 6 mgr per liter of bacterial culture, and as we obtainedthe soluble protein at a concentration of 6 mgr/ml, it is clear that theexpression is comparable or better than other scFv's or FAB's expressedin E. coli. Furthermore, the solubility of the camel V_(HH) αTT2 iscertainly better than that obtained for the mouse V_(H) fragments. Theyield and solubility is certainly in the range needed for mostapplications.

To prove the proper folding of the purified protein, the αTT2 wasbrought at a concentration of 3.9×10⁻⁶ M and used it for CD measurement(FIG. 7). The CD spectrum is characteristic for a polypeptide with aβ-pleated sheet folding as expected for a well structured immunoglobulinfold (Johnson, 1990).

The Camel Anti-Tetanus Toxoid V_(HH) Affinity Measurements

The binding of the camel V_(HH) antibody to the tetanus toxoidimmobilised on the microtiter plates was revealed by the successiveincubation with firstly, the rabbit anti-camel V_(HH) or rabbitanti-dromedary IgG, and secondly, a goat anti-rabbit/alkalinephosphatase conjugated antibodies (Sigma). The apparent affinity of thecamel V_(HH) proteins against tetanus toxoid was estimated by inhibitionELISA exactly as described by Persson et al. (1991) for the humananti-tetanus toxoid FAB fragments they produced in E. coli.

The specificity of the soluble camel V_(HH) for the tetanus toxoid wassuggested from the ELISA experiments in which we competed the bindingwith free antigen was competed. An apparent inhibition constant ofaround 10⁻⁷, 10⁻⁸ M was observed for both V_(HH) fragments (FIG. 8).This compares favorably with the inhibition constants for the humananti-tetanus toxoid FAB fragments cloned by Persson et al. (1991) whichwere in the range of 10⁻⁷ to 10⁻⁹ M.

The measurement of the affinity constant by ELISA is however, morereliable if determined according to the procedure of Friguet et al.(1987). With this protocol we found an affinity constant of 6.10⁷ M⁻¹and 2.10⁷ M⁻¹ for the αTT1 and αTT2 respectively. These affinities areconsistent with a specific V_(HH)-antigen interaction (the polyspecificantibodies generally bind their antigen with affinities of 10⁶ M⁻¹ orless (Casali et al. 1989)).

Epitope Recognition of αTT1 and αTT2.

Tetanus toxin consists of three domains. The C fragment binds to theneuronal cells, it is said to be the neurospecific binding domain. The Bdomain appears to be involved in the neuronal penetration of the Adomain or L chain (Montecucco & Schiavo, 1993). The L chain isresponsible for the intracellular activity.

The C fragment is the most immunogenic part of the tetanus neurotoxin,and a recombinant C fragment is commercially available (Boehringer andCalbiochem). We showed by ELISA that the αTT1 bacterial extract bindsequally well both to the complete tetanus toxoid and to the recombinantC fragment. Therefore the epitope of this camel V_(HH) is present on theC fragment. By contrast, the αTT2 extract binds to the complete tetanustoxoid, but not to the C fragment. Therefore the αTT2 recognizes anepitope located on the A or B domain.

The in vivo Neutralization of Tetanus Toxin Toxicity

The neutralizing activity of the purified camel αTT1 or αTT2 V_(HH)domains against tetanus toxin was tested. As a control, eight NMRI miceof 8 to 12 weeks (80 to 100 gr) were injected I.P. with 400 ng tetanustoxin (SmithKline Beecham Biologicals) (=10 times the LD50) in 0.1 mlPBS. To test the neutralizing activity of the camel V_(HH) αTT1 or αTT2we preincubated 4 or 40 mg of this purified recombinant protein with 400ng of the tetanus toxin in 0.1 ml of PBS for 30 minutes before I.P.injection into the mice. The survival of the mice was followed over aperiod of 2 weeks (FIG. 9). It is clear that all mice injected with thetetanus toxin alone or in the presence of a non-specific purified camelV_(HH) (cVH21 of Muyldermans et al., 1994) were killed within 3 days.The survival of the mice injected with the tetanus toxin was increasedsignificantly by the co-injection of only 4 mg of the purified camelαTT1 or αTT2. The survival was even more pronounced for the co-injectionof tetanus toxin with 40 mg of camel V_(HH). It appears that the αTT1had a slightly higher neutralizing activity than the αTT2. This couldoriginate from its intrinsic higher affinity for binding the tetanustoxin (Simpson et al., 1990). Alternatively it might result from thebinding of the αTT1 V_(HH) to the fragment C of the tetanus toxin whichinhibits more the toxic effect than the binding of the αTT2 to itsepitope outside the C fragment.

EXAMPLE II

Generation of Specific Camel V_(HH) Fragments Against Lysozyme

Using the same protocol as the one described in Example I (specificsteps or conditions modifying those of Example I are indicatedhereafter) for the generation of specific camel V_(HH) fragments havinga specificity and an affinity for tetanus toxoid, V_(HH) fragments havebeen obtained against lysozyme.

We chose the Hen Egg Lysozyme (HEL) as an antigen to immunize a camel(Camelus dromedarius). This protein was selected for the reason thatcomparisons can be made with several other mouse monoclonal antibodyfragments recognizing the same antigen and of which the structure evenin complex with its antigen are known.

Camel Immunization

The serum of a camel was shown to be non-reacting with lysozyme. Weinjected this camel with 100 μg lysozyme (Boehringer) at days 9, 30, 52,90 and with 50 μg at days 220, 293 and 449. The blood was collected onaverage 3 days after each injection.

The following steps were then performed as in Example 1.

mRNA purification of camel blood lymphocytes.

cDNA synthesis and PCR amplification of camel V_(HH) gene.

Construction of Camel V_(HH) library.

Panning with lysozyme (the Falcon 3046′ plates were coated with 1 mg/mllysozyme).

96 colonies were randomly chosen and grown individually in LB medium.

The virions were prepared and tested for their binding activity againstlysozyme immobilised on microtiter plates.

The percentage of binders was increasing after each round of panning.Twenty positive clones were grown and tested by PCR to check thepresence of an insert with the proper size of the V_(HH) gene, and theirDNA was finally sequenced. The sequencing data revealed that twodifferent clones were present among this set of 10 clones. The plasmidDNA of these clones was named pHEN4-αLYS2 and pHEN4-αLYS3, and it wasshown that these two different clones contained a cDNA coding for acamel V_(HH) (FIGS. 11, 12). Comparison of the amino acids in theseclones with the camel V_(HH) clones we analysed before (Muyldermans etal., 1994) clearly indicated that the anti-lysozyme camel V_(HH)originated from a heavy chain immunoglobulin lacking the CH1 domain andlight chains. Especially the identity of the key residues at position 11(Ser), 37 (Phe), 44 (Glu), 45 (Arg) and 47 (Gly) proved this statement(Muyldermans et al., 1994).

Production of Soluble Camel V_(HH) with Anti-Lysozyme Activity

For the purification of the anti-lysozyme camel V_(HH) we concentratedthe periplasmic extract 10 times by ultrafiltration (Millipore membranewith a cut off of 5000 Da). After filtration the concentrated extractfrom the pHEN4-αLYS2 can be purified by Protein A-Sepharosechromatography. Elution of the αLYS2 V_(HH) is done with 100 mMTri-ethanol amine. The pH of eluate is immediately neutralized with 1 MTris-HCl (pH 7.4). Unfortunately the expressed αLYS3 V_(HH) does notbind to Protein A. Therefore the purification has to be performed byaffinity chromatography. The concentrated extract is applied on a columnof lysozyme immobilized on CNBr-Sepharose (Pharmacia). Elution of theanti-lysozyme V_(HH) is obtained with 100 mM Tri-ethanolamine. Theeluate has to be neutralized as described above. Further purification ofboth anti-lysozyme V_(HH)'S can obtained by gel filtration onSuperdex-75 (Pharmacia) equilibrated with PBS (10 mM phosphate buffer pH7.2, 150 mM NaCl). The peak containing the anti-lysozyme activity elutedat the expected molecular weight of 16,000 Da indicating that theprotein behaved as a monomer and doesn't dimerize in solution. Thefractions containing the pure V_(HH) (as determined by SDS-PAGE) werepooled and the concentration was measured spectrophotometrically. Ayield of 5 mg of purified protein per liter of bacterial culture wascalculated. The purified protein could be further concentrated byultrafiltration to 10 mg/ml in PBS or water without any sign ofaggregation, as seen on the UV spectrum.

The Camel Anti-Lysozyme V_(HH) Affinity Measurements

The specificity of the soluble camel V_(HH) for the lysozyme wassuggested from the ELISA experiments in which we competed the bindingwith free antigen. An apparent inhibition constant of around 5.10⁻⁷ and5.10⁻⁸ M was observed for the α-LYS3 and α-LYS2 respectively. Theseaffinities are consistent with a specific V_(HH)-antigen interaction(the polyspecific antibodies generally bind their antigen withaffinities of 10⁶ M⁻¹ or less (Casali et al. 1989).

Epitope Recognition of α-LYS2 and α-LYS3

To analyse whether the two camel V_(HH) with anti-lysozyme activity bindto the same or to different epitopes we used the techniques of additivebinding in ELISA (Friguet et al., 1989). An addivity index of more than40 indicates pairs of antibodies that can bind simultaneously on theantigen, while addivity indices of less than 20 is characteristic forpairs of antibodies with overlapping epitopes. Our camel α-LYS2 andα-LYS3 had an addivity index of 45. From this experiment it appears thatthe α-LYS2 and α-LYS3 bind to different epitopes on the lysozymemolecule.

EXAMPLE 3

Making Bivalent Monospecific or Monovalent Bispecific Binding Constructsfrom Camelid V_(HH)'s

From the camel V_(HH)'s with specificity to tetanus toxin (α-TT1 orα-TT2) or with specificity to lysozyme (α-LYS2 or α-LYS3) cloned in thepHEN4 bacterial expression vector, we made constructs with followingcharacteristics:

1. V_(HH) with ProX repeat sequences of the camel long hinge includingthe 3 Cys and part of the CH2 domain. These constructs can be also usedas an intermediate for the next constructs.

2. V_(HH) with ProX repeat sequences of the long hinge of camel with oneCys followed by a stopcodon in the pHEN4. These are bivalent constructswith monospecificity.

3. V_(HH) linked with the ProX repeat sequences of the long hinge ofcamel (without Cys) followed by a second V_(HH). These are monovalentconstructs with bispecificity, or bivalent constructs withmonospecificity depending on the V_(HH)'s.

1. Camel V_(HH) with Camel Long Hinge and Dart of CH2 Domain

The (pHEN4-α-LYS3) or the (pHEN4-α-TT2) plasmids were digested withBstEII and Xmn I. BstEII cuts in the framework 4 of the camel V_(HH),and Xmn I cuts in the β-lactamase gene of pHEN4. The DNA fragmentcontaining the camel V_(HH) was isolated from agarose gel.

A clone containing a camel V_(HH) with unknown specificity, the camellong hinge and the first part of the CH2 domain cloned in pBluescript(Statagene) was cut with the same enzymes (Bst EII and Xmn I) and theDNA resulting fragment containing the hinge and CH2 parts was isolatedfrom agarose gel.

The two DNA fragments (one containing the V_(HH) of determinedspecificity, the other containing the coding sequence of the hinge andCH₂ domains) were mixed and ligated to each other and used to transformE. coli cells. As a result a (pHEN4-α-LYS3-long hinge-CH2) plasmid and a(pHEN4-α-TT2-long hinge-CH2) plasmid have been obtained.

2. Bivalent Monospecific Constructs (FIGS. 13A, 13B and 15)

The (pHEN4-α-LYS3-long hinge-CH2) plasmid was taken as template foramplification with primers A4 and AM007 (SEQ ID NOS: 9 & 10,respectively).

A4 (Sfi I Site Underlined):5′-CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCGA(G,T)GT(G, C)CAGCT-3′

AM007: 5′-GGCCATTTGCGGCCGCATTCCATGGGTTCAGGTTTTGG-3′

These primers anneal respectively with their 3′ end to the beginning ofthe V_(HH) and to the end of the structural upper hinge of the camellong hinge sequence. The primer AM007 will extend the 3′ end of theα-LYS3 or of the α-TT2 gene (depending on the template) with (SEQ ID NO:12) CCCATGGAATGCGGCCGCAAATGTCC. The NcoI and NotI sites are underlined.These nucleotides up to the Not I site code for the amino acids Pro MetGlu Cys.

The PCR fragment is double digested with Sfi I and Not I, and theresulting fragments are cloned in the pHEN4 vector cleaved with the sameenzymes. The ligated material is transformed in WK6 E. coli cells andselected on ampicillin. The transformed clones are checked for theirinsert by PCR and by sequencing. The plasmid (pHEN4-α-LYS3 longhinge/Cys) and (pHEN4-α-TT2-long hinge/Cys) were generated.

The extraction of the expressed V_(HH) α-LYS3-long hinge/Cys orα-TT2-long hinge/Cys proteins lead to isolation of a dimerised moleculebecause of the formation of the disulfide bridge between the Cys residuewithin the long hinge. Both camel V_(HH) dimer constructs (α-LYS3 longhinge/Cys)₂ and α-TT2 long hinge/Cys)₂ are well expressed in E coli uponinduction with IPTG, and are easily obtained from the periplasm. Theywere quite soluble and bound the original antigen with high affinity andhigh specificity.

3. Monovalent Bispecific Protein Constructs (FIGS. 14A, 14B and 16)

In the previous plasmid constructs (pHEN4-α-LYS3 long hinge/Cys) and(pHEN4-α-TT2-long hinge/Cys), we have two restriction sites for Nco I.Digestion of the plasmid with this enzyme allows the isolation of thecamel V_(HH) gene followed by the long hinge without the Cys codon.Ligation of the (α-LYS3-long hinge) fragment into the pHEN4-α-LYS2 or inthe pHEN4-α-TT2 plasmids linearised with Nco I creates the plasmids(pHEN4-α-LYS3-long hinge linker-.alpha.-LYS2) or (pHEN4-.alpha.-LYS3longhinge linker-α-TT2). Expression of the gene leads to the production ofthe α-LYS3 V_(HH) linked to the α-LYS2 V_(HH) or linked to the α-TT2V_(HH) by the intermediate of a linker based on the structural upperhinge of the camel long hinge.

Following this protocol monovalent bispecific proteins consisting of thecamel V_(HH) of α-LYS3 linked to the camel V_(HH) of α-LYS2 and that ofthe camel V_(HH) of α-LYS3 linked to the camel V_(HH) of α-TT2 can beisolated. Both proteins are expressed well in E coli and can beextracted from the periplasm. In ELISA the binding properties of thelatter protein to the tetanus toxoid and to the lysosyme can be shown.

With these gene constructs at hand it becomes possible andstraightforward to exchange either V_(HH) with any other V_(HH) withanother specificity.

For example we can exchange the second camel V_(HH) by digesting theplasmid with Pst I, or with Nco I and to ligate the DNA fragmentcontaining the V_(HH)-long hinge linker into the pHEN4-V_(HH) linearisedwith either Pst I or Nco I.

Similarly, we exchanged the first camel V_(HH) α-LYS3 gene from the(pHEN4-α-LYS3 long hinge linker-α-LYS2) plasmid construct into(pHEN4-α-TT1-long hinge linker-α-LYS2). This was done by cutting theplasmid with BstEII and further ligating the DNA fragment containing the(long hinge linker-α-LYS2) into the (pHEN4-α-TT1) plasmid linearisedwith BstEII.

With a slight modification of this protocol it becomes even possible togenerate multivalent constructs. In this case the (V_(HH)-long hingelinker-V_(HH)) plasmid needs to be digested with BstEII and the DNAfragment containing the (long hinge linker-V_(HH)) gene should beisolated from agarose gel. Because of the asymmetry in the recognitionsite of BstEII, it is only possible to obtain head-to-tail ligationsupon self ligation. The self-ligated DNA should thereafter (with orwithout prior size selection) be ligated into the pHEN4-V_(HH) plasmidlinearised with BstEII. This will create a plasmid of the type(pHEN4-[V_(HH)-long hinge linker]_(n)).

REFERENCES CITED IN THE PRECEDING EXAMPLES

-   Borrebaeck et al., (1992) Bio/Technology 10, 697-698.-   Casali et al., (1989) Annu. Rev. Immunol. 7, 513-536.-   Friguet et al., (1983) J.Immunol. Meth. 60, 351 et (1989) Protein    Structure.-   A practical approach (Ed. T. E. Creighton) IRL Press p. 287-310)-   Glockshuber et al., (1990) Biochemistry 29, 1362-1367.-   Hamers-Casterman et al., (1993) Nature 363, 446-448.-   Hoogenboom et al., (1991) Nucl. Acids Res. 19, 4133-4137.-   Johnson W. C., (1990) Proteins: Structure, Func & Genetics 7,    205-214.-   Kabat et a. (1991) Sequences of Proteins of Immunological Interest    (US Dept.Health Human Services, Washington) 5th Ed.-   Marks et al., (1991) J. Mol. Biol. 222, 581-597.-   Montecucco & Schiavo (1993) TIBS 18, 324-329.-   Mullinax et al., (1990) Proc.Natl.Acad.Sci USA 87, 8095-8099.-   Persson et al., (1991) Proc.Natl.Acad.Sci USA 88, 2432-2436.-   Sambrook et al., (1989) Molecular Cloning CSHL Press-   Simpson et al., (1990) J. Pharmacol. & Exp. Therap. 254, 98-103.-   Skerra and Pluckthun, (1988) Science 240, 1038-1040.    Sequence CWU 1

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1. A method for treating a subject comprising the step of passiveimmunization or immunotherapy with a variable fragment of a heavypolypeptide chain (V_(HH)) of an immunoglobulin devoid of light chains.2. The method according to claim 1, wherein the passive immunization isa treatment for infection or intoxication due to a bacterial toxin. 3.The method according to claim 2, wherein the intoxication is acuteintoxication or intoxication due to drug resistant bacteria.
 4. Themethod according to claim 2, wherein the infection or intoxication isdue to infection by bacterial agents or viral agents.
 5. The methodaccording to claim 4, wherein the bacterial agent is Clostridium,Staphylococcus, Pseudomonas, Pasteurella, Yersinia, Bacillus anthracis,Neisseria, Vibrio, E. coli, Salmonella, Shigella, Listeria,Corynebacterium diphtheriae or a pathogen ingested with food.
 6. Themethod according to claim 5, wherein the Clostridium is Clostridiumtetani, Clostridium botulinum or Clostridium perfringens.
 7. The methodaccording to claim 5, wherein the Vibrio is Vibrio cholera.
 8. Themethod according to claim 5, wherein the E. coli is enterotoxic E. coli.9. The method according to claim 2, wherein the intoxication is due tobites or contact with a toxic invertebrate or toxic vertebrate.
 10. Themethod according to claim 9, wherein the toxic invertebrate is a seaanemone, a coral, a jellyfish, a spider, a bee, a wasp or a scorpion.11. The method according to claim 9, wherein the vertebrate is avenomous snake.
 12. The method according to claim 11, wherein thevenomous snake is a snake of the Viperidae family, Crotolidae family orLapidae family.
 13. The method according to claim 1, wherein thetreatment is for tetanus, botulism, gangrene, necrotic enteritis,enterotoxemia, food poisoning, ocular Psuedomonas infection, diphtheria,or anthrax.
 14. The method according to claim 1, wherein the passiveimmunization or immunotherapy is performed in combination with medicalor surgical use of a neurotoxin.
 15. The method according to claim 14,wherein the neurotoxin is botulinum toxin.
 16. The method according toclaim 1, wherein the subject is a human.